The objectives of this grant application is to develop novel methods of antigen presentation that enhances the success of monoclonal antibody (mAb) production to small molecules including haptens and peptides. mAbs are nature's biological weapons, able to target and help eliminate disease-causing agents from the human body. Though it is relatively easy to produce specific high affinity mAbs to exogenous proteins, it remains a challenge to obtain high affinity and specific mAbs to small molecules. Major reasons are a) immunological tolerance (clonal suppression, clonal anergy, immunological silencing, or clonal deletion of specific clones), b) repertoire reshaping and receptor editing, c) inability of the small molecules to elicit strong Ab response due to simplicity of the molecular structure and/or short half life of the molecule in vivo, d) the steric hindrance of the small molecule by protein carrier and e) immunogenicity of the protein carrier. Our preliminary results using the plant-derived cadiotonic steroid-like digitalis glycoside, ouabain (Qua), indicate that the last 2 mechanisms are plausible and therefore we chose to test them. To overcome problems associated with protein carrier immunogenicity, we propose to immunize mice with either homologous proteins or protein variants that exhibit minor structural differences from the host proteins and to avoid problems associated with steric hindrance we propose to incorporate spacers between the hapten and the protein carrier. Our hypothesis is that by integrating these 2 strategies, the protein structure will be modified to the extent that it can be recognized by the immune cells and yet will not be as immunogenic as the exogenous proteins and therefore the Ab response will be directed towards the hapten and hence high affinity and highly specific mAbs can be elicited. As a model small molecule, Oua will be used and as a model protein carrier, A/J or Balb/c derived immunoglobulins and variants of host proteins such as cytochrome C and lysozyme will be utilized. Spacers include either crosslinkers or different length peptides. A/J or Balb/c mouse strain will be used for immunization and mAb production. Success in this work would enhance our ability to produce high affinity mAbs to small molecules and to extend them to produce high affinity mAbs to peptides. Such mAbs are useful molecular probes and can be used for detection, purification and functional analysis of the small molecules and peptides. [unreadable] [unreadable]